Development of polarized plasma membrane domains is a prerequisite for renal epithelial function and its perturbation contributes to a variey of pathologies. Thus, elucidating the mechanisms through which polarity is generated and maintained is fundamental to developing a thorough understanding of renal physiology. Epithelial cells avail themselves of a complex sorting apparatus to create and maintain the compositional integrity of their plasma membrane domains. During the previous funding period of this award we have employed new approaches that have illuminated aspects of the sorting and signaling systems involved in generating and maintaining renal epithelial cell polarity. Our previous efforts focused in part upon the sorting and trafficking properties of the Na,K-ATPase, the basolateral ion pump that generates the driving force for the vast majority of renal fluid and electrolyte transport. A number of recent studies suggest that the Na,K-ATPase is not simply an occupant of the basolateral cell surface, but is in addition an active participant in the intercelllar communication and signaling pathways that establish the composition and structure of the basolateral plasma membrane domain. In the present application we propose to apply innovative tools to investigate further the pathways and partners involved in the sorting and delivery of apical and basolateral membrane proteins. We will carry out studies that explore novel hypotheses relating to both the cellular mechanisms responsible for epithelial polarity and to their physiological implications. Towards this end we will 1) Define pathways pursued by newly synthesized proteins as they travel to the apical and basolateral domains of the plasma membrane; 2) Determine how sorting and trafficking behaviors vary among renal epithelial cell types and during the course of renal development; and 3) Identify and characterize temporal and polarity-dependent interactomes. Through this work we will develop new insights into the mechanisms that mediate membrane protein trafficking in renal epithelial cells and that therefore determine the physiological properties of the renal tubule.